Diverse, uncultivated bacteria and archaea underlying the cycling of dissolved protein in the ocean

16 Pages·2016·0.85 MB·English

by  William D Orsi

TheISMEJournal(2016)10,2158–2173
©2016InternationalSocietyforMicrobialEcology Allrightsreserved 1751-7362/16
www.nature.com/ismej
ORIGINAL ARTICLE
Diverse, uncultivated bacteria and archaea
underlying the cycling of dissolved protein in
the ocean
William D Orsi1,7, Jason M Smith2, Shuting Liu3, Zhanfei Liu3, Carole M Sakamoto2,
Susanne Wilken2, Camille Poirier2, Thomas A Richards4,5, Patrick J Keeling5,6,
Alexandra Z Worden2,5 and Alyson E Santoro1,5
1Horn Point Laboratory, University of Maryland Center for Environmental Science, Cambridge, MD, USA;
2Monterey Bay Aquarium Research Institute, Moss Landing, CA, USA; 3Department of Marine Science,
UniversityofTexasatAustin,PortAransas,TX,USA;4DepartmentofBiosciences,UniversityofExeter,Exeter,
UK; 5Integrated Microbial Biodiversity Program, Canadian Institute for Advanced Research, Toronto, Ontario,
Canada and 6Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada
Dissolvedorganicnitrogen(DON)supportsasignificantamountofheterotrophicproductionintheocean.
Yet,todate,theidentityanddiversityofmicrobialgroupsthattransformDONarenotwellunderstood.To
better understand the organisms responsible for transforming high molecular weight (HMW)-DON in the
upper ocean, isotopically labeled protein extract from Micromonas pusilla, a eukaryotic member of the
resident phytoplankton community, was added as substrate to euphotic zone water from the central
CaliforniaCurrentsystem.Carbonandnitrogenremineralizationratesfromtheaddedproteinsrangedfrom
0.002to0.35μmolCl−1perdayand0.03to0.27nmolNl−1perday.DNAstable-isotopeprobing(DNA-SIP)
coupled with high-throughput sequencing of 16S rRNA genes linked the activityof 77 uncultivated free-
livingandparticle-associatedbacterialandarchaealtaxatotheutilizationofMicromonasproteinextract.
The high-throughput DNA-SIP method was sensitive in detecting isotopic assimilation by individual
operational taxonomic units (OTUs), as substrate assimilation was observed after only 24h. Many
uncultivated free-living microbial taxa are newly implicated in the cycling of dissolved proteins affiliated
with the Verrucomicrobia, Planctomycetes, Actinobacteria and Marine Group II (MGII) Euryarchaeota. In
addition,aparticle-associatedcommunityactivelycyclingDONwasdiscovered,dominatedbyuncultivated
organisms affiliated with MGII, Flavobacteria, Planctomycetes, Verrucomicrobia and Bdellovibrionaceae.
The number of taxa assimilating protein correlated with genomic representation of TonB-dependent
receptor(TBDR)-encodinggenes,suggestingapossibleroleofTBDRinutilizationofdissolvedproteinsby
marinemicrobes.Ourresultssignificantlyexpandtheknownmicrobialdiversitymediatingthecyclingof
dissolvedproteinsintheocean.
TheISME Journal (2016) 10,2158–2173; doi:10.1038/ismej.2016.20; publishedonline8March 2016
Introduction molecular weight (LMW, HMW) compounds and
compound classes of varying concentration and
Dissolved organic nitrogen (DON) is the second most
bioavailabilityincludingaminoacids,N-acetylamino
abundant form of fixed nitrogen in the ocean, with
polysaccharides (for example, chitin and peptidogly-
a pool size of 7.7 (±2.3)×104 Tg N (Karl et al., 2001;
can),dissolvedproteinsanduncharacterizableprotei-
Bronk,2002;AluwihareandMeador,2009).Thetotal
naceous matter (Tanoue et al., 1995; McCarthy et al.,
DON pool consists of numerous low and high
1997,1998;Aluwihareetal.,2005).Microbialhetero-
trophy can control the flux and composition of DON
(Carlson and Ducklow, 1995), yet the specific organ-
Correspondence:WDOrsi,DepartmentofEarthandEnvironmental isms responsible for DON transformations and their
Sciences,Lüdwig-MaximiliansUniversität,München,Germany. biochemical mechanisms are poorly understood
E-mail:[e-mail]
(Aluwihare and Meador, 2009).
or AE Santoro, Horn Point Laboratory, University of Maryland
Phytoplankton are a major DON source (Nguyen
CenterforEnvironmentalScience,Cambridge,MD,USA.
E-mail:[e-mail] and Harvey, 1997; Bronk and Steinberg, 2009).
7Current address: Department of Earth and Environmental Release of DON results from a variety of forms
Sciences, Lüdwig-Maximilians Universität München, München, of phytoplankton cell death including viral lysis
Germany.
(Fuhrman, 1999) and grazing (Worden et al., 2015),
Received 21 July 2015; revised 14 January 2016; accepted
18January2016;publishedonline8March2016 and can also be released via passive diffusion
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(Fogg, 1971). The liberated dissolved proteins unexplored. Several of the aforementioned groups,
and oligopeptides are initially regarded as labile including the MGII, have been found in association
HMW-DON compounds (Keil and Kirchman, 1993; with particulate organic matter (Delong et al., 1993),
Keil et al., 2000), but become more refractory where they have an enhanced genomic capacity for
because of processes such as abiotic complexation HMW substrate utilization (Orsi et al., 2015).
with existing dissolved organic matter (DOM) (Keil We sought to determine whether microbial taxa
and Kirchman, 1994). Dissolved proteins are previouslyimplicatedinHWM-DOMturnovermight
recycled faster relative to the bulk HMW-DON specifically be involved in HMW-DON (protein)
pool, as evidenced by their lower δ15N values cycling. We further hypothesized that MGII archaea
(Meadoretal.,2007)andmoremodernΔ14Cvalues are actively involved in protein turnover, and that
(Loh et al., 2004) relative to bulk HMW-DON. this activity would be enhanced in association
Protein in dissolved and particulate fractions with particles (Orsi et al., 2015). To this end,
can be readily degraded in seawater by microbial we performed DNA stable-isotope probing (SIP)
activity(SizemoreandStevenson,1974;Smithetal., using isotopically labeled (13C and 15N) proteins
1982),andturnoverratesofproteinscanbethesame from the picoeukaryotic phytoplankter Micromonas
order of magnitude as turnover rates of dissolved pusilla. We coupled SIP with high-throughput
free amino acids (DFAA) (Hollibaugh and Azam, Illumina sequencing of 16S small subunit ribosomal
1983).LMW-DONsuchasDFAAreleasedbyprotein RNA (rRNA) genes (Hungate et al., 2015; Morando
hydrolysis (Billen and Fontigny, 1987) are readily and Capone, in review) in three different size
assimilated by bacteria in aquatic systems (Hobbie fractions to identify protein assimilation by indivi-
etal.,1968)andcansupportupto~50%ofbacterial dual bacterial and archaeal operational taxonomic
production in the oceans (Kirchman, 2000). units (OTUs). We performed the incubations at
StudieslinkingsomemicrobialgroupstoLMW-DON a station within the coastal transition zone of
(Alderkampetal.,2006; Nikradetal.,2012; Liu etal., the Central California Current, where there is a rich
2013) and HMW-DON (Nagata et al., 1998; Cottrell contextual background on biogeochemistry (for
and Kirchman, 2000) uptake have demonstrated example, Collins et al., 2003), and where M. pusilla
important spatiotemporal variability in DON utiliza- is a resident of the phytoplankton community
tion, but have been limited in their ability to under- (Thomsen and Buck, 1998). Free-living (0.2–0.8μm)
standthefulldiversityofbacteriaandarchaeainvolved and particle-associated (43μm) fractions contained
in DON cycling by the need for a priori selection adiversityof newly implicated, uncultivated bacter-
oftargetgroups.Nevertheless,arelativelylargenumber ial and archaeal taxa mediating the cycling of
of bacterial taxa in the ocean have been implicated in dissolved protein. Our results suggest that when
HWM-DOM turnover, either through experimental dissolved proteins become available, highly diverse
additions or following phytoplankton blooms, and microbial populations with the genomic capacity
someoverlapshouldbeexpectedbetweenthesetaxa totransportHMW-DONcanacquireitrapidly.These
and those involved in HWM-DON cycling. These results refine our understanding of DOM turnover
include the Alteromonads (for example, McCarren in the ocean and indicate a strong affinity of
et al., 2010; Sharma et al., 2014; Mayali et al., 2015), several uncultivated clades for dissolved protein,
Flavobacteria (for example, Kirchman, 2002; Rinta- and suggest that diverse particle-attached microbial
Kanto et al., 2012; Teeling et al., 2012; Sharma et al., communities can act as a DON sink.
2014; Mayali et al., 2015) and the gammaproteobac-
terial clades NOR5/OM60, SAR92 (Teeling et al.,
2012; Sharma et al., 2014) and SAR86 (Dupont et al., Methods
2012). Bacterial groups specifically implicated in
the cycling of HMW-DON and proteins include Sampling
the Flavobacteria (Cottrell and Kirchman, 2000; Seawater was collected aboard the R/V Western Flyer
Kirchman, 2002), the γ-Proteobacteria subgroups during Monterey Bay Aquarium Research Institute
Arctic96B16, Ant4D3 and SAR86 (Nikrad et al., cruise CN13ID, 7–17 October 2013, along California
2014), and the α-Proteobacteria clade SAR11 Cooperative Fisheries Investigations (CalCOFI) Line 67
(Malmstrom et al., 2005). (Collinsetal.,2003;Penningtonetal.,2010).Sampling
The uncultivated Marine Group II Euryarchaeaota and hydrographic profiling were conducted using
(MGII) are also predicted to have a role in DON a conductivity-temperature-depth rosette sampler
transformations, based on the recovery of protein (Sea-Bird Electronics) equipped with (12) 10l Niskin
degradation pathways in metagenomic data (Iverson samplebottles.Inadditiontothestandardconductivity-
etal.,2012;Orsietal.,2015).MGIIarethoughttobe temperature-depth measurements, the profiling rosette
heterotrophs, through their utilization of LMW-DON was equipped with the following sensors: WETstar
(Alderkamp et al., 2006) and available metagenomic fluorometer (Wetlabs, Philomath, OR, USA), transmiss-
data(Frigaardetal.,2006;Zhangetal.,2015).However, ometer (SeaTech, Bellevue, WA, USA), SBE43 dis-
direct evidence of their role in utilizing dissolved solved oxygen sensor (Sea-Bird Electronics, Bellevue,
proteins has not been demonstrated, and interactions WA, USA) and a radiometer (Biospherical, Inc.,
between archaea and HWM organic matter are largely San Diego, CA, USA). Chlorophyll a (Chl a) pigment
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concentrations were determined by fluorometry using shore,withinthecoastaltransitionzonebetweenthe
established protocols (Pennington and Chavez, 2000). coastal upwelling and oligotrophic waters offshore
Major phytoplankton groups (Prochlorococcus, Syne- (Collins et al., 2003). The site was selected because
chococcus and picoeukaryotic phytoplankton) were of the historic presence of a significant picoeukar-
quantified at sea by flow cytometry as described yote population M. pusilla (Thomsen and Buck,
previously (Cuvelier et al., 2010). 1998), and previous characterization of particle-
associated taxa (Orsi et al., 2015). Water for the SIP
incubationwasacquiredfrom20mdepth.M.pusilla
Stable isotope probing incubation proteinextractwasaddedto4lofseawatertoafinal
M. pusilla CCMP1545wasgrownfor10daysat21°C concentration of 28μgl−1 in trace metal-clean poly-
under a light:dark cycle of 14h:10h using cool white carbonate bottles. Bottles were amended with dual
light (150μmol Q m−2 s−1) in L1 medium (Guillard isotope-labeled proteins (13C and 15N), unlabeled
and Hargraves, 1993) based on artificial seawater proteins or no proteins, in triplicate. Bottles were
amended with 2.5mM 99 atm% 13C-labeled sodium incubated for 24h in the dark in coolers that were
bicarbonate and 882μM 98 atm% 15N-labeled sodium halfway filled with surface seawater to maintain
nitrate (Cambridge Isotope Laboratories, Andover, in situ temperature. The bottles were subsampled
MA, USA). The growth rate was 0.47 per day (s.d.: every 4h (except for the 16h timepoint) to determine
0.09).Isotopicenrichmentofthecellswasobtainedby the concentration of ammonium and DFAA, and
mixing the culture in unlabeled media 1:1 with the nitrogen isotopic composition of NO−+NO−
3 2
labeled medium, incubating for 2 days, followed by (δ15N ). The carbon isotopic composition of
NOx
another 1:1 mixture with labeled media. This was dissolved inorganic carbon (DIC) was determined in
repeated five times. The isotopic composition of samplestakenat24h(seedetailsbelow).Attheendof
whole cells was measured at the UC Davis Stable the incubation 1–2l of seawater was filtered sequen-
Isotope Facility using online combustion coupled to tially through 3μm (Pall Versapor-3000T, Port
amassspectrometer;valueswere16atm%for13Cand Washington, NY, USA), 0.8μm (Pall Supor-800, Port
42atm% for 15N. It should be noted, however, that Washington, NY, USA) and 0.2μm (Pall Supor-200,
particulateCisotopiccompositionwasdeterminedfor Port Washington, NY, USA) filters using a peristaltic
cellsthathadbeeninstationaryphaseforseveraldays pump,andDNAwasextractedaspreviouslydescribed
relative to the cells harvested for protein extraction, (Santoro et al., 2010). Catalyzed reporter deposition—
potentially leading to an underestimation of the fluorescence in situ hybridization for Archaea and
starting atm% 13C. Euryarchaeota, DAPI staining and epifluorescence
Proteins from M. pusilla were extracted according microscopy followed the protocol of Orsi et al.
to the method by Santoro et al., 2015. In brief, cell (2015). This protocol utilized the Eury806 probe that
pellets were incubated at room temperature for isspecificforEuryarchaeota(Tieraetal.,2004)andthe
15min in an extraction buffer, followed by heating Arch915probethattargetsthedomainArchaea(Stahl
at 95°C for 10min and shaking at 350r.p.m. for 1h. and Amann, 1991), but also binds non-specifically to
Protein extract was removed into a new sterile tube some pelagic marine bacteria (Pernthaler et al., 2002).
and centrifuged for 30min at 4000r.p.m. at room
temperature, followed by filtration through a 5-μm
pore size sterile filter. Filtered samples were then Ammonium, DFAA and DCAA measurements
concentrated using 6ml 5000 MWCO Vivaspin Water column [NH+] samples were collected in
4
columns (Sartorius, Göttingen, Germany) down to 125ml acid-cleaned polyethylene bottles and incu-
400μl, split in half, and precipitated with 1.6ml of bation samples were collected in 27ml acid-cleaned
cold (4°C) solution of 1:1 MeOH and acetone, and polyethylene vials and refrigerated. Samples were
0.5mM HCl for 3 days at −20°C. Precipitated analyzed within hours using a fluorometric method
samples were spun down for 30min at 4°C at with a single working reagent (orthophthaldialde-
14500r.p.m., air dried in a laminar flow hood and hyde, sodium sulfite and sodium borate) (Kerouel
resuspended in 1ml extraction buffer prior to and Aminot, 1997; Holmes et al., 1999). The system
quantification. Protein extracts were quantified was automated with a peristaltic pump-based flow
fluorometrically. No DNA was measureable in the injection analysis. Detection limits, estimated as 1%
extractsusingfluorometricmeasurements (detection of full scale, gave +/− 3nM with the high sensitivity
limit=2ngμl−1) suggesting that co-extraction of injection loop. An estimate of the precision from
nucleicacidswiththeproteinswasminor.However, a cast where samples were analyzed from 12 bottles
we cannot exclude the possibility that a small tripped at the same location gave 25 +/− 1nM.
amount of labeled nucleic acids were co-extracted Depth profiles of DFAA andtotal dissolved amino
with the proteins. There were no visible particles in acids (TDAA) were collected from the rosette into
the extracts suggesting that the protein was mostly pre-combusted (450°C, 5h) glass scintillation vials
dissolved. afterfilteringthroughasterile0.2-μmporesizefilter
Incubation experiments (SIP and remineralization and analyzed as described below. DFAA were
rates) were conducted at CalCOFI station 67–70 analyzed by high performance liquid chromatogra-
(36.74 °N, 122.02 °W), approximately 170 km from phy (Shimadzu Prominence, Kyoto, Japan) with
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fluorescence detection after pre-column o-phthal- was measured in acidified samples using a GasBench
dialdehyde derivatization (Lee et al., 2000). TDAA IIsysteminterfacedtoaDeltaVPLUSisotoperatiomass
were analyzed in the same way as DFAA but after spectrometer (ThermoFinnigan) at the UC Davis
hydrolysis by 6N HCl under nitrogen at 110°C Stable Isotope Facility. δ15N was measured and
NOx
for 20h (Kuznetsova and Lee, 2002). Dissolved rates of remineralization calculated as described in
combined amino acids (DCAA) were calculated the previous section. A range of initial isotopic
as the difference between TDAA and DFAA. The enrichment values for the starting 13C atm% enrich-
coefficient of variation for replicate DFAA and ment in the labeled proteins was used in the rate
DCAA samples was 10–20%. The concentrations calculations,becauseofpotentialeffectsofthedelayin
of individual amino acids were calculated on the measuringthestarting13Catm%oftheM.pusillacells
basis of an amino acid external standard mixture (see above). We used a lower value of 16 atm% 13C
(Amino Acid Standard H, Thermo Scientific, based on the whole cell measurement and an upper
Waltham, MA, USA). valueof95atm%13Cbasedonthecalculatedatm%13C
forthenumberofcelldoublingsthatoccurredassuming
uptake of fully labeled medium. The starting ambient
In situ array DCAA pool was assumed to have a δ13C of −20‰
Water for measurements of 13C, 15N-labeled protein (McCarthyetal.,2004)andδ15Nof8‰(McCarthyetal.,
remineralization and 15NH+ oxidation rates was 2007). Limitations to this method include an incom-
4
collected from depths of 2, 5, 10, 20, 30 and 50m plete characterization of both the isotopically labeled
usingtheconductivity-temperature-depthrosettesam- protein preparation and the ambient DCAA pool, and
pling system described above. Upon return to deck, considerableuncertaintiesinthestartingconcentrations
water from each depth was drained directly into of both (Lipschultz, 2008).
clear, acid-cleaned, 280ml polycarbonate bottles.
Following addition of isotopically labeled substrates
to a final concentration of 50nmoll−1 and 28μgl−1
Density gradient centrifugation and gradient
for 15NH4+ and 13C, 15N-proteins, respectively, the fractionation
incubation bottles were loaded into hand-made Atthe24htimepoint,oneofthetriplicateexperimental
nylon mesh bags, hung at their depth of origin on (13C, 15N protein addition) and control (unlabeled
a 6.3mm double-braided line attached to a custom protein addition) bottles (see above) was sampled for
GPS-enabled Surface Temperature Experimental DNA-SIP. DNA was extracted from the experimental
Longitude Latitude Asset (STELLA) drifter (http:// and control bottles after 24h according to the method
www.mbari.org/bog/drifterdata/)witha60cmsurface of Santoro et al. (2010). In brief, 850μl of lysis buffer
float, and deployed in situ from dusk to dawn and 100μl of 10% SDS were added to 2ml gasketed
(ca. 12h). Following recovery of the array, samples bead-beating tubes containing the filters and 0.1mm
were immediately filtered through combusted GF/F sterileglassbeads(Biospec,Kyoto,Japan).Beadbeating
filters housed in Swinnex filter holders (EMD Milli- wasperformedfor1minandsamplesheatedfor2min
pore, Billerica, MA, USA), into acid-washed 60ml at99°C.Afterheating,25μlof20mgml−1proteinaseK
HDPE bottles and kept frozen until analysis. The was added and tubes were incubated over night at
δ15NNOx was determined at the University of 55°C.DNA was thenpurifiedfromthelysate withthe
ConnecticutAveryPointcampususingthedenitrifier DNeasy Blood and Tissue Kit (Qiagen, Venlo,
method (Sigman et al., 2001) and a GasBench and Netherlands).
PreCon trace gas concentration system interfaced to Theprotocolfordensitygradientcentrifugationand
a Delta VPLUS mass spectrometer (ThermoFinnigan, gradient fractionation followed previously described
Bremen,Germany).Nitrateisotopereferencematerials
methods for DNA-SIP (Neufeld et al., 2007; Dunford
USGS32, USGS34 and USGS35 were analyzed in
and Neufeld, 2010), with some minor modifications.
parallel to calibrate δ15N values. δ15N precision,
Inbrief,densitygradientcentrifugationwasperformed
determined by repeat analysis of the reference
in a TV90 vertical rotor at 20°C for 40h at 177000g
materials, was found to be 0.15‰ for the entire data
inanOptimaXL-90ultracentrifuge(BeckmanCoulter,
set.Resultantδ15N datawereusedtocalculaterates
NOx Brea, CA, USA). DNA was spun in 4ml polyallomer
of NH+ oxidation or labeled protein remineralization
4 Optiseal tubes (Beckman Coulter) in cesium
using previously described methods (Smith et al.,
chloride (CsCl) gradients with an average density of
2014) derived from the original equations laid out by
1.725gml−1. Centrifuged gradients were fractionated
Dugdale and Goering (1967).
into 15 equal fractions via careful manual pipetting
from the top of the polyallomer tube. DNA was
Protein remineralization rates precipitated with 2 volumes of polyethylene glycol
Protein-derived C and N remineralization rates were for2hatroomtemperatureandpelletedbycentrifuga-
determined in bottle incubations on the in situ array tion(30min,13000g).Pelletswerewashedoncewith
andtheon-deckSIPexperimentbymeasuringchanges 70% ethanol, resuspended in molecular grade water,
in stable-isotope ratios in the DIC (δ13C-DIC) and and quantified fluorometrically using a Qubit (Life
inorganic nitrogen (δ15N ) pools after 24h. δ13C-DIC Technologies, Carlsbad, CA, USA).
NOx
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PCR and qPCR relatively unimodal distribution in the CsCl gradi-
16S rRNA genes from each fraction were amplified ent and whose peak buoyant density (BD) was
using primers equipped for dual-indexed barcoded shifted 40.012gml−1 between the experiment
sequencingonanIllumina(SanDiego,CA,USA)MiSeq and the control (similar to Buckley et al., 2007a).
(Kozich et al., 2013), which target the V4 hypervari- In this case, a relatively higher number of
ableregion(515F/806R;(Caporasoetal.,2012)).We sequences from an OTU retrieved in the heavy part
used a version of the 515F primer with a single-base ofthegradientcomparedwithitsdistributioninthe
modification (in bold) to increase coverage of the control is a function of isotopically labeled protein
SAR11 clade (515F: 5′ – GTGCCAGCMGCCGCGGT assimilation. Thus, throughout the manuscript, we
AA – 3′; Apprill et al., 2015). PCR reactions were refer to OTUs meeting the criteria defined above
carried out in 25μl volumes with 2μl of template as those assimilating protein.
added to each reaction for 35 cycles. Each gradient
fraction was amplified using a different barcode
combination and amplicons were gel-extracted Results
(QIAquick Kit, Qiagen) and pooled in equimolar
Cruise setting and vertical biogeochemical profiles
concentrations prior to high-throughput paired-end
Oceanographic conditions during the cruise were
Illumina sequencing (MiSeq, 2×250bp reads) at the
characterized by a general decreasing gradient in
University of Illinois Keck Center for Comparative
phytoplankton biomass with distance from shore,
andFunctionalGenomics.Eachdensityfractionwas
with upwelling and higher concentrations of Chl a
also screened for the major subgroups of marine
alongthecoast(SupplementaryFigureS1).Thesite
archaea using quantitative PCR (qPCR). qPCR
for the DNA-SIP incubations, station 67–70, had
for MGII Euryarchaeaota followed a previously
lower surface Chl a concentrations compared with
published assay (Orsi et al., 2015), while the qPCR
neighboring stations to the east and west (1.5–2
assayforMarineGroupIammonia-oxidizingarchaea
versus 43μgl −1) (Supplementary Figure S1) and
using the ammonia monooxygenase subunit A
exhibitedabroadsubsurfacechlorophyllmaximum
(amoA) gene followed that of Santoro et al. (2010).
spanning the depths of 17–40m (Figure 1a).
The phytoplankton community (quantified by flow
cytometry) was dominated by Prochlorococcus
Bioinformatic analysis
(48% of phytoplankton cells) and Synechococcus
Processing of the paired-end MiSeq data was per-
(37% of phytoplankton cells). Eukaryotic phyto-
formed according to Kozich et al. (2013) using
plankton comprised 15% of total phytoplankton
MOTHUR (Schloss et al., 2009), and pair-wise OTU
cells (Supplementary Figure S2). NH+ concentra-
clustering was performed in QIIME (Caporaso et al., 4
2010). A total of 12.5 million quality-checked 16S tions ranged from below detection limits to 260nM,
rRNA gene sequences corresponding to 2722 OTUs with a maximum of 260nM at 45m (Figure 1a).
Vertical profiles of DFAA and TDAA (the combina-
were used for downstream analysis. Sequences were
tionofDFAAandpeptides)at67–70showapeakin
clustered at 97% sequence identity using UCLUST
DFAA and TDAA at 40m (Figure 1b), which
(Edgar, 2010). OTUs were identified to taxonomic
coincides with the subsurface NH+ maximum at
groups through BLASTn searches against the SILVA 4
40m(Figure1a).Concentrationsofmostindividual
database(Pruesseetal.,2007).OTUtableswererarified
DFAA peak at 40m and progressively decrease
per size fraction, to the sample with the least number
of sequences (0.2–0.8μm=27,327; 0.8–3μm=57,110; in deeper water (down to 500m), with threonine,
43μm: 34,970). Rarified counts for each OTU in valine, beta-alanine and methionine being excep-
tions to this trend (Supplementary Figure S3). The
the control and SIP gradients were normalized to the
composition of DFAA between 0–40m is signifi-
maximal abundance of that OTU across density
cantly different from the DFAA pool found in
fractions in unlabeled or SIP-labeled bottles. Only
OTUs with 410 sequences that were detected in waters 60–500m (analysis of similarity: P=0.001).
450% of the fractions were used in downstream Protein remineralization rates from the in situ
array ranged from 0.002 to 0.35μmolCl−1 per day
analysis. After these quality control criteria, a total of
with a maximum rate at 20m (Figure 1c). The
4.1million16SrRNAgenesequencescorrespondingto
maximum protein C remineralization rate at 20m
702 OTUs were used for downstream analysis.
Sequence data have been deposited in the NCBI Short coincideswiththesubsurfacechlorophyllmaximum
Read Archive under BioProject ID PRJNA287804. (Figure 1a) and was the depth sampled for the SIP
We defined DNA-SIP incorporation similar to experiment. The protein N remineralization rates
NelsonandCarlson(2012)andHungateetal.(2015) (ammonification plus nitrification) measured on the
on the basis of a comparison of the relative in situ array ranged from 0.03 to 0.27nmol N l−1
distribution of an OTU across the CsCl density per day, with a maximal rate at 5m. Protein N
gradient in a control (12C and 14N substrate added) remineralization rates (±s.d.) in the SIP incubation
versus an experimental (13C and 15N substrate were similar to those measured on the array under
added) incubation. OTUs incorporating the label in situ light conditions (0.11 versus 0.14nmol N l−1
were defined as those OTUs that exhibited a per day. In comparison, protein C remineralization
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0 0
0
m)
h ( 50 50 50
pt
e
D
100 100 100
0 1 2 3 0 50 100 150 200 0 0.1 0.2 0.3
Chl a (µg L-1) DAA (nM) µmol C L-1d-1
DFAA TDAA
0 50 100 150 200 250 0 0.2 0.4 30
[NH+ ] nM nmol N L-1d-1
4
Protein C Protein N
remineralizationremineralization Nitrification
Figure1 (a)VerticalprofilesofChlaandNH+atstation67–70,errorbarsrepresents.d.ofChlameasurementsovera3-dayperiodat
4
station67–70.(b)VerticalprofilesofDFAAandTDAAatstation67–70.(c)InsituremineralizationratesofproteinCandN,andNH+
4
oxidationrates.ProteinNremineralizationratesaretheresultofammonificationplusnitrification.
rates in the SIP incubation were slightly higher than dominated by Verrucomicrobia and the Flavobacteria
the rates measured on the array under in situ light (Supplementary Figure S7). The most abundant
conditions (0.55 (±0.09) μmol C l−1 per day). NH+ free-living taxa were affiliated with the SAR11 clade
4
oxidation rates ranged from 0.2nmol l−1 per day followed by the SAR86 clade (Figure 2), whereas the
atthesurfacetoamaximumof29.8nmoll−1perday dominant taxa in the particle-associated size fractions
at 50m (Figure 1c). were affiliated with Synechococcus, Roseibacillus,
Fluviicola, Formosa and uncultivated Flavobacteria
(Figures 2). There was a shift in the BD of peak DNA
Timecourse measurements quantity between the experimental and control bottles
There was no significant difference in net NH+ in all size fractions (Supplementary Figures S8A–C).
4
production between control (no protein added) and Moreover, the taxonomic composition of density frac-
experimental (protein added) bottles at 0 and 24h tions varied significantly as a function of BD between
(t test: P40.05) (Supplementary Figures S4A and B). experimental and control bottles (analysis of similar-
Furthermore, there was no increase in DFAA concen- ity: Po0.05; Supplementary Figures S8D–F). No
tration after 24h between bottles that did, or did discernable difference in community structure was
not, receive proteins. Depletion of aspartate, glycine, observed between control (no protein added) and
histidine,alanine,valine,serineandleucinewasfaster experimental (protein added) incubations after 24h
in bottles amended with proteins, relative to una- (Supplementary Figure S7). Post incubation, commu-
mended control (Supplementary Figure S5). There nities in the 0.8–3μm and 43μm fractions were
were no significant differences in total free-living similar to each other and contained relatively higher
DAPI-stainedcellsbetweentreatmentsafter24h(ttest: percentages of Flavobacteria, Planctomycetes and
P40.05; Supplementary Figure S4C). Total DAPI- Verrucomicrobia compared with free-living fractions
stainedcellsattachedtoparticlesalsodidnotincrease (Supplementary Figure S7).
significantly over the course of the experiment (t test; Seventy-seven bacterial and archaeal OTUs
P40.05; Supplementary Figure S4D). However, free- assimilated the amended protein after 24h
living euryarchaea increased significantly after 24h (Supplementary Table S1), which exhibit high
(Supplementary Figure S4C). Interestingly, no archaea variability in relative abundance (Table 1). OTUs
were observed on diatomaceaous particles, whereas affiliated with γ-Proteobacteria (SAR86, KI89A,
ahighnumberofbacteriawerefoundattachedtothese OM60 and SAR92 clades), Verrucomicrobia, Flavo-
particles (Supplementary Figure S6). bacteriaandMGIIhadthehighestnumbersofOTUs
assimilating protein, and had the highest percen-
tages of OTUs utilizing proteins compared with
Free-living and particle-attached bacteria and archaea other groups (Supplementary Table S1). The domi-
utilizing dissolved proteins nant taxa in the free-living fraction assimilating
The microbial community in the free-living fraction protein were affiliated with the α-Proteobacteria
was dominated by the α-Proteobacteria and γ-Proteo- (Roseobacter, Rhodobacteraceae), γ-Proteobacteria
bacteria, whereas the particle-associated fractions were (SAR86clade),andMGII(Figure2).Therewaslittle
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Percent of total reads Percent of total reads Percent of total reads
in 0.2 - 0.8 µm in 0.8 - 3 µm in >3 µm
0 4 8 12 0 2 4 6 8 10 0 1 2 3 4 5 6
SAR11 clade Synechococcus sp. Fluviicola sp. *
SAR86 clade NS7 marine group Synechococcus sp.
SAR86 clade * SAR86 clade Roseibacillus sp. *
OCS155 marine group Fluviicola sp. NS7 marine group
SAR11 clade Roseibacillus sp. Formosa sp.
SAR86 clade Formosa sp. Flavobacteriaceae sp.
SAR11 clade Flavobacteriaceae sp. NS4 marine group *
AEGEAN-169 group Prochlorococcus sp. OM27 clade
AEGEAN-169 group Roseobacter clade OCT SAR86 clade
NS5 marine group SAR92 clade Roseibacillus sp.
OCS116 clade Marine Group II Cloacibacterium sp.
NS5 marine group NS4 marine group NS9 marine group *
SAR86 clade NS9 marine group * Achromobacter sp.
Rhodobacteraceae * SAR86 clade NS9 marine group *
Roseobacter clade OCT* NS11-12 marine group Owenweeksia sp. *
SAR406 clade NS9 marine group SAR92 clade
Marinicella sp. Rhodobacteraceae Chitinophagaceae sp.
Marine Group II Marinoscillum sp. Puniceicoccaceae sp.
NS4 marine group Owenweeksia sp. NS9 marine group *
Pseudospirillum sp.* Nereida sp. NS11-12 marine group
NNSS77 F mmlKuaaIvrr8iiii9nncAeeo l ggacrrl oo asuudpppe.* N SM5 Sa mrAinaRer1i nG1e6r o gcurlopaud IpeI S yR nMAoeslcacehraiiobnncaievoc oGcilrclrauuoxssu p sss pppII... *
SAR11 clade Sufflavibacter sp. SAR86 clade
Sufflavibacter sp. DEV007 Marine Group II
OM60(NOR5) clade* OM60(NOR5) clade Fluviicola sp.
Marine Group II* SAR86 clade DEV007
Verrucomicrobia Gammaproteobacteria Deltaproteobacteria Betaproteobacteria Euryarchaeota Flavobacteria
Cyanobacteria Alphaproteobacteria Planctomycetes Marine group A Actinobacteria
Figure 2 Relative abundance of the most abundant OTUs in each of the three size fractions after 24h, asterisks indicate OTUs that
assimilatedprotein.
overlap in protein-utilizing taxa between the size Discussion
fractions (Figure 3). All of the Bacteroidetes-
Phytoplankton biomass supports significant second-
affiliated OTUs assimilating protein belonged to
ary production by heterotrophic prokaryotes, and
the Flavobacteria. One SAR86 OTU that assimi-
is central to organic matter cycling in the ocean.
lated protein was the third most abundant OTU
Phytoplankton-derived proteins likely have an
(Figure 2) detected in the free-living fraction
important role in this process. Here, we investigated
(Figure 4) and was affiliated with the SAR86 clade
the specific microbial taxa involved in the uptake
III (Figure 5b).
of dissolved proteins, both on and off particles, and
Microbial populations that assimilated protein in
found that although some protein-assimilating taxa
particle-associated size fractions are markedly dif-
were previously identified in the utilization of
ferent from free-living populations. For example,
phytoplankton-derived organic matter, others are
OTUs affiliated with Planctomycetes, Fluviicola (Fla-
newly implicated.
vobacteria), Formosa (Flavobacteria), Roseibacillus
(Verrucomicrobia) and OM27 clade (δ-Proteobacteria) As expected, we detected protein assimilation
by free-living α-Proteobacteria, γ-Proteobacteria and
were the dominant populations assimilating proteins
Flavobacteria previously implicated in this process.
in the particle-attached fractions (Table 1, Supple-
The relative percentage of α-Proteobacteria assimilat-
mentary Table S1). Most of these bacterial taxa were
ing protein (o1%, Table 1) is markedly lower than
not observed to assimilate protein in the free-living
observed in a study from coastal waters, where 13%
fraction and were not abundant in the free-living
ofα-Proteobacteriacellsassimilatedprotein(Cottrell
fraction (Figure 2), although some were detected in
and Kirchman, 2000). In contrast, roughly 6% of
relativelylowabundance(datanotshown).Moreover,
totalSAR86(γ-Proteobacteria)sequencesassimilated
marineEuryarchaeotaaffiliatedwiththeMGII.Aclade
assimilated protein in both the 0.2–0.8 and 0.8–3μm protein in the free-living fraction (Supplementary
Table S1), which is comparable with a MAR-FISH
size fractions, whereas the MGII.C clade only assimi-
lated protein in the 0.2–0.8μm fraction (Figure 5a). study that found 8% of all SAR86 cells assimilated
The assimilation of protein by marine euryarchaea protein in the surface ocean (Nikrad et al., 2014).
was further validated by qPCR (Figure 6a). MGII Methodological differences preclude a direct com-
incubated with labeled protein exhibit higher quan- parison between the fraction of labeled cells by
titiesofrRNAgenesingradientfractions41.71gml−1 MAR-FISH and the fraction of a clade with SIP-
ineverysizefractionrelativetothecontrol(Figure6a). labeled OTUs (carried out here), but the indirect
This assimilation of protein by heterotrophic euryarch- comparison here shows that the same groups are
aea was markedly larger compared with the ammonia- found to assimilate protein with both methods.
oxidizing thaumarchaea, which did not meet our The ability of SAR86 to acquire protein may be
criteria for proteinassimilation (Figure 6b). facilitated by their genomic capacity for HMW
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BDshift%totalertaxon 41%46%000.1%2%10%2%0 erstothe salaufftbielsditarmtaetodereawcpiqtruhoitsetihitnieotnhSaA(nDRua9np2yoncolttahedeteraO(lF.,Ti2gUu01r(Fe2i)g4.u)Areans4sO,imTBUDi-
p ref shift;0.036gml−1),suggestingthatthisclademaybe
sal %)%) 1%)4%)%)%) %) ’otal important for protein recycling in areas where the
ncetot 134.3 0.00.00.30.1 18 %t SAR92 clade reaches high abundance, such as
e% ((00((((0( ‘ coastal waters (Stingl et al., 2007). Flavobacterial
u 70 2714 4 e
4μ3m Seqwith 105523388 12352,0468 14225 BD.Th cDbeuOltlNsin(hCaoovutertreoalplpearnnefdoecrKeeinarncchesmtfuoardny,H,2Ma00Wl0o;-wDKeOirrNcphemorvcaeennr,tL2agM0e0W2o)-f,
n
TUswithBDshift%total) 6(2.4%)8(1.2%)002(0.3%)1(0.2%)3(0.4%)2(0.3%)01(4.6%) ndshiftsi fpcroaemree-pdliavwrienidtgh oFwtlhiatvehrobgtarhocetueprpisae(rTcaaesbnslitemag1iel)a,tweodfhicpFhrlaoivtseoaibnlasocctoelormwia-
O ( 1 3 a assimilating protein in coastal waters (Cottrell and
e
Table1Asummaryoftaxaassimilatingproteinafterthe24hincubation –μ–μMicrobialgroup0.20.8m0.83m AverageAverageSequenceswithBDshiftSequencesBDshiftOTUswithOTUswithAverageBDshiftBDshift%total%totalwith%total%totalBDshiftBDshiftBDshift−−−111(gml(gmlpertaxonpertaxon(%total)(%total))))(gml Flavobacteria0.0182(0.3%)319(0.04%)0.3%0.0279(1.2%)29035(2%)3%0.020Verrucomicrobia0.0163(0.4%)2524(0.34%)51%0.0184(0.5%)18835(1.3%)10%0.014Euryarchaeota(MGII)0.0145(0.6%)9776(1.3%)52%0.0121(0.2%)704(0.05%)0.6%0Gammaproteobacteria0.023(0.5%)44354(6%)22%0.0174(0.5%)315(0.02%)0.07%0Alphaproteobacteria0.0162(0.3%)5755(0.8%)2%0.0143(0.4%)79(0.005%)0.01%0.012Actinobacteria0.0121(0.2%)350(0.04%)0.7%00000.012Planctomycetes00000.0212(0.3%)1678(0.11%)10%0.01Deltaproteobacteria00000.0166(0.3%)4619(0.3%)21%0.012Cyanobacteria00000.0181(0.3%)21(0.001%)0.006%0Total16(3%)63078(8%)30(3.7%)55286(3.8%) Abbreviations:BD,buoyantdensity;OTU,operationaltaxonomicunit.ForallOTUsexhibitingaBDshift,seeSupplementaryTableS1forabundanc‘’percentoftotalsequencesorpercentoftotalOTUspersizefraction.%totalpertaxonreferstopercentoftotalsequencespertaxonomicgroup. 12doruabFsdppOpmacbKPpbFpTFFaatFtOwaapcsisPCsoheiiilflssrs0f9eaallleirrrlrlairtarhzziaTTofaaaaeuustvaarssgiseooouWOWoreccc0m9rrieetdoilodsnnpiijicvvUUluevtvvhttttonmtdsttio29mmimuitieeeueiceheeezerooooiiefimnfraacetecsr))sriisoiiirrtpirrgitehdmbbnniiistlccussiaaonnnglaneanoiilltu(ephlahtfeieaaesecoosdfaacrmaaaylSlsasrooagda5effsoat-satccttadtllnattotiitfuuapisurnftueilocaameciiytdttnr(bliitonoe(ynoasdwoneeettcTipldtocnh)an,y,ncTsiiaintonagtiadfpfgtrrnotgtnmlrepnatiycciea2aiodtolaaetrisiinaalxtohrts.th.weclrguaastylO0uay,bwidrf(hetuoidttieHeneaaTidfochislgpdll0unnstlOnFaetnTsad1mepcltdoi.dlehmaaftaeieMr0aptsdasgtittnel9hsocnrUurohionbwbveiedaoiuupt)lT.efpwasle9n,saunensf.iO1rltgrWuranitdisngvrrisieoo9eyrbgcaotzllrs1s)tieosttnioegIhiTe(obtttii).entcaoashriviniv-F9icauhbn.nenh1tnndcestaecUdDoRotdvhtteipore9aWsesit,eriktiuhisitacooAoumrayefaroiWoesOgtaeebirlrcmr5ussalntllutlSsnitunrorvtnnnmnadtrtieiaheaessdn)ecerlsMtsuetztT2avhmep,v,uetrtetao-detheektaisisebprai6eiid(o(onraasnhbungtianotiaginnpnwtS7btfyoenbptd2acsbfnbnieminsdnahffhugiafoo.cayumctelxoimltanni4teripcdeeeerdctl(perrbwteapm(eamcditaiO%etu,daidpmizahetraBhiaohanaranhAsentpieoctliltt-niieiniStpe'ncc4teporeahe-stpeehenGcsSsntlttrecteislttrtl1srlniemdueensa‘oaecvhiruiueedtieitinospei(o3vom)hrmci(reheaioatnctgftAanDrepsls,nRme,aenor4lieillnslvteliaaebμk.vilOinevterefeceaiasbdsoaaipr,ttmvip(orayevmeaLdgnp-t,cr0zea.emtigo4avTbeaeicathmizadnawpetgoadTi.cdotacaenua1egsnar8ThttUaraPneenatdnaortftnhdeltrnhco%isitseiliimreamfettlμrauolgsmptpiroclltsdw’iiraysibeteb(artrhcciyeymzncuaielHs1nrcrn;Heubiiaattlceealmhfraelooncacipe0iphaacefTcloelc-tiMessotaoFsetlotrtteap%aatdodl-ltTresroh-ieeoaeohtanlSlldae.a-sssScaaotrfitWrisatf.l,uaniibaesmulyhrgmre1t,o)rshetitdnnp)nv.xstotaiblavtb-ih,snsF)attvao2w2eea-eadaaslrvya,o.ceaaccisiiotltaDbtii1tflao0v.ll0c,icw0aeectfiictettdgvniulaTiaci9airtlfttiS1hheOax0ame9vhzholhiiievhatehottoaegAtt9hrnlit2oo5r1neee9ehheheennMaiiieeotrhnltlle1aa3ssidnd)nndnddndddn))o6yya.geeeessssr.t--ltt,,,,..;,
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assimilatedproteincomparedwithmostothermicrobial phytoplankton-derived substrates, such as the diatom
groups,anddidnotassimilateproteininthefree-living cellwallproteinsilaffin(ZeiglerAllenetal.,2012).The
fraction (Table 1). Utilization of M. pusilla proteins by successofplanctomycetecellsonparticlesmaybedue
planctomycetes is consistent with their previous detec- to their unique holdfast structures and stalks, which
tion following phytoplankton blooms (Morris et al., allow them to attach to surfaces (Fuerst, 1995). Most
2006) and metagenomic evidence indicating that Planctomycetes OTUs that assimilated protein were
metabolism of marine planctomycetes is linked to affiliatedwiththeCL500–3clade(SupplementaryTable
S1), an as-yet uncultured subgroup found in oligo-
trophiclakes(Urbachetal.,2001).AnotherPlanctomy-
0.2 - 0.8 µm cete-affiliated OTU that assimilated protein in the
0.8 - 3 µm 43μm fraction was affiliated with the uncultivated
Urania-1B-19 sediment group (Supplementary Table
S1), reportedfromdeepsea sedimentof the Mediterra-
32
nean (Heijset al., 2008).
4 34
The discovery of protein utilization by a diversity
of uncultivated Verrucomicrobia taxa in all size
2
5 fractions (Supplementary Table S1) suggests that
VerrucomicrobiaareimportantforDONcyclingboth
onandoffofparticles.Therelativelyhighproportion
of Verrucomicrobia taxa assimilating protein in the
42 free-living (51%) and 43μm fractions (46%) indi-
cates that they have a relatively strong preference for
dissolved proteins compared with most other groups
detected (Table 1). In the free-living fraction, Verruco-
>3 µm microbia protein utilization was dominated by repre-
sentatives of the Puniceicoccaceae, previously shown
Figure3 Thenumberofoverlappingandnon-overlappingOTUs
to assimilate exudates from Synechococcus in eupho-
between size fractions that assimilated protein. Note that most
OTUsassimilatedproteininonlyonesizefraction. tic zone waters (Nelson and Carlson, 2012). This
0.2 - 0.8 µm 0.8 - 3 µm >3 µm
SAR92 Unc. Puniceicoccaceae Unc. Puniceicoccaceae Unc. Roseibacillus sp. Unc. Fluviicola sp NS9
100
0
MGII.C Unc. Rhodobacteraceae NS9 CL500 NS9 Unc Owenweeksia
100
e
c
an 0
d
un SAR86 Unc. Rhodobacteraceae OM27 OM27 Unc. Fluviicola sp Unc. Fluviicola sp
ab100
m
u
m
xi
a
m 0
of
nt SAR92 MGII.A Unc. Formosa sp. Unc. Flavobacteriaceae Unc. Roseibacillus sp OM27
ce100
er
P
0
MGII.A Unc. Puniceicoccaceae CL500 unc Flavobacteriaceae Unc. Formosa sp NS2b
100
0
1.70 1.76 1.70 1.76 1.70 1.76 1.70 1.76 1.70 1.76 1.70 1.76
CsCl density (g ml-1)
Verrucomicrobia Gammaproteobacteria Deltaproteobacteria Flavobacteria
Euryarchaeota Alphaproteobacteria Planctomycetes
Figure4 OTUsexhibitingthegreatestdegreeofproteinassimilation.LinesrepresentthedistributionofOTUsinthecontrol(unlabeled
proteins,dashedlines)andexperiment(labeledproteins,solidlines).TheyaxisrepresentstherelativeabundanceofeachOTUnormalized
toitsmaximalabundanceacrossalldensityfractions.SeeSupplementaryTableS1forrelativeabundanceandBDshiftsforallOTUs.
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14757605210497927 14334262 333206745826601772 NS2b
73710324180479514 1144333344224500 Clade I 255928674 NS5
374723508 MGII.C 14334247
145651477 99662623575442 NS4
56790779 2707393 NS10
145651471 34112904 148732632
31143341874982905 304835044663588663 Clade II 236828281435261416
189909252
6049054 170784128 148729979
383933299 304694082 326485912
298716312 304658374 260071296401068018 NS9
298716312 304658179 307567362
170516806 17225773 148732320
31737408241808064 MGII.A 1 923628690309779441122 151314398037733572296473664
197313284 222427968
58615802 8575489 Clade III 148732618
47079393 14313940276045 1 79 259124035315361793 NS7
93116005 151933607
47079393 148732512 315361570
57639640 MGII.B 0.02 19174345293920615
0.1 193084194 320153420 Fluviicola
62958780
62958781
0.2 - 0.8 µm 0.8 - 3 µm > 3 µm Fluviicola taffensis
62958778
189172435
283463638
170283880 NS11-12
197311348
0.05
Figure 5 Maximum likelihood phylogenies (PhyML) of MGII (a), SAR86 (b) and Flavobacteria (c) 16S rRNA genes sharing 497%
sequenceidentitywithOTUs,whichassimilated(redfont),ordidnotassimilate(boldblackfont),proteinaftera24hincubation.Colored
squaresindicatethesizefractionwhereproteinassimilationwasobserved.Blackcirclesatnodesrepresentbootstrapsupportof490%
andwhitecirclesrepresentbootstrapsupportof450%.NumbersrepresentNCBIGIaccessionIDs.
supportsbiogeographicandgenomicevidencesuggest- divide inside the periplasmic space causing the prey
ing that certain marine Verrucomicrobia species have cell size to increase (Abram et al., 1974). This cross-
a preference for phytoplankton-derived HMW-DOM feeding phenomenon of predatory bacteria has been
(Herleman et al., 2013). Verrucomicrobia have also observed in previous DNA-SIP studies, whereby
been identified as degraders of polysaccharides in the Bdellovibrio populations assimilated C after preying
Arctic (Cardman et al., 2014) and given their ubiquity on methanotrophs incubated with 13C methane
in the ocean and presence on POM (Freitas et al., (Morrisetal.,2002).WehypothesizethatOM27clade
2014),chemoheterotrophicactivityofVerrucomicrobia organisms correspond to an uncultivated group of
taxa should contribute markedly to DOM and POM predatorybacteriabecause(i)theclosestphylogenetic
turnover. OTUs affiliated with Roseibacillus were the relation of this clade is to the predatory Bdellovibrio
dominantverrucomicrobialgroupassimilatingprotein genus (Fuchs et al., 2005) and (ii) 1–2μm-sized
in the particle-associated fraction (Supplementary bdelloplasts (Fenton et al., 2010) should be enriched
Table S1). The few cultivated Roseibacillus species in the 0.8–3μm size range, which is the size range
do not exhibit N-acetyl-β-glucosaminidase, trypsin where OM27 group-specific protein assimilation
or chymotrypsin activity (Yoon et al., 2008), enzymes (21%) was higher than all other groups (Table 1).
whichdegradeHMW-DONsuchaschitinandprotein. WeconcludethatOM27cladeorganismsacquiredthe
Thus, it seems likely that some particle-attached label through predation on bacteria that had assimi-
Roseibacillus cells might instead acquire protein C lated the dissolved protein substrate (that is, cross-
and N by transporting the oligopeptides released feeding), and were then captured as swollen bdello-
throughproteinhydrolysisfromextracellularenzymes plasts in the 0.8–3μm fraction.
manufactured by other bacteria. We confirmed protein utilization by uncultivated
Protein assimilation was identified in OM27 clade taxa affiliated with marine Euryarchaeota (Figure 5a).
organisms(SupplementaryTableS1),whicharemost Our study provides the firstdirect link betweenMGII
closely related to the predatory deltaproteobacterial cells and protein recycling, as well as identification
genus Bdellovibrio (Fuchs et al., 2005) and have of the specific MGII taxa utilizing dissolved proteins.
a geographically wide distribution (for example, Compared with the other major microbial groups,
Rappe et al., 1997; Fuchs et al., 2005). The OM27 a relativelyhigh percentage (52%)of free-living MGII
clade had the highest percentage of OTUs (21%) OTUs assimilated protein, suggesting that they have
assimilating protein compared to all other groups in a strong affinity for this substrate (Table 1). The
the 0.8–3μm fraction suggesting their activity was physiology of planktonic MGII is hypothesized to
relatively high in this size range (Table 1). This may be based on protein metabolism (Reysenbach and
indicate the formation and activity of bdelloplasts, Flores, 2008; Iverson et al., 2012), and heterotrophic
structures that are formed as predatory Bdellovibrio euryarchaea in sediments also appear to utilize
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